【作者】 何桥；

【导师】 梁国鲁； 高中山；

【作者基本信息】 西南大学 ， 果树学， 2010， 博士

【摘要】 枇杷(Eriobotrya japonica Lindl.)原产于我国,其栽培历史悠久,是我国亚热带地区的珍稀特产水果,具有较高的经济价值。浙江和江苏是我国著名的枇杷主产区,拥有丰富的枇杷种质资源,也属于高度进化枇杷类型栽培区。因此,以当地枇杷主栽品种或类型为主,开展我国主要枇杷品种的遗传多样性和亲缘关系研究,对枇杷种质资源的有效保护和创新利用具有重要的指导意义。本论文以收集到的浙江和江苏主栽枇杷品种或类型为主,结合福建和四川主栽品种,以及栎叶枇杷和大渡河枇杷共54个品种或类型为试材。选取在苹果和枇杷遗传连锁图谱上已定位的110个苹果SSR标记,筛选可用于枇杷遗传分析的SSR引物,建立枇杷SSR标记体系,对54个供试材料进行遗传多样性分析和品种鉴别,并对大五星天然三倍体枇杷株系的遗传多样性进行了研究,筛选可用于天然三倍体枇杷鉴定的SSR标记,主要结果如下：1、建立了枇杷SSR反应体系,利用‘鸡蛋红’、‘夹脚’、‘长红’、‘铜皮’、‘大五星’、‘早黄’、‘大红袍’和‘大玫瑰红袍’8个品种,从110对SSR引物中筛选出在8个枇杷品种中具有多态性的引物78对,多态性引物比率为71%,通过梯度PCR分析,筛选出78对引物的适宜退火温度。2、利用39对SSR引物对54个供试材料进行遗传多样性分析,共扩增出155个等位基因。AT000400-SSR、CH01h02、CH03c02和CH01d03四对引物为多基因座引物,不用于遗传多样性分析。在不统计大渡河枇杷和栎叶枇杷等位基因的情况下,各引物52个栽培枇杷的等位基因数为2-7个,平均每对引物扩增出3.38个等位基因。有效等位基因数最高的是引物CH03a09,有4.01个等位基因,CH01f02有效等位基因数最少,为1.04,35对引物的平均有效等位基因数为2.21。香农指数最小的是CH01f02,为0.10,CH03a09最高,为1.49,平均香农指数为0.84。观察杂合度为0—0.83,CH01f02最低,Hi08a04最高,平均为0.47；期望杂合度最高的是CH03a09和CH02d12,均为0.76；CH01f02最低,为0.04；平均期望杂合度为0.50。固定指数从Hi08a04的-0.71到CH01f02的1.00,平均为0.07,表明供试枇杷种质间具有丰富的遗传多样性。对供试材料进行UPGMA聚类分析,以SM相似性系数0.48为阀值,将供试材料分成栽培枇杷和野生枇杷两组。栽培枇杷以0.723的SM相似性系数为阀值,可分成A、B和C三个亚组。A亚组包括所有浙江和江苏地方类型,地理来源很近,属温带类型,来自四川的‘龙泉1号’、福建的‘长红’以及西班牙的两个品种’Marc’和‘Peluches’也聚在A亚组内。B亚组主要是福建品种‘早钟6号’、‘解放钟’、‘香钟’、‘太城4号’和‘白梨’,属于亚热带品种,日本品种‘森尾早生’作为早钟6号的亲本之一,也聚在该亚组；此外,四川栽培品种‘金丰’也聚在B亚组内。C亚组仅由四川栽培品种‘大五星’组成。主成分分析结果与UPGMA聚类分析结果基本一致,两个野生枇杷类型与栽培枇杷类型明显区分开,栽培类型主要根据其地理分布分成4组,第Ⅰ组包括所有浙江类型和10个江苏类型,日本枇杷品种‘森尾早生’及西班牙品种’Marc’、’Peluches’;第Ⅱ组包含15个江苏枇杷类型；第Ⅲ组由6个福建枇杷品种组成；四川主栽品种‘大五星’和‘龙泉1号’组成第Ⅳ组。39对多态性SSR引物可区分’Marc’与’Peluches’两个西班牙品种及‘美玉’与‘常绿2号’两个江苏品种和类型外的所有供试枇杷类型。SSR基因型分析结果验证了‘早钟6号’(解放钟×森尾早生)、‘常绿5号’(白玉×甜种)和‘香钟’的亲缘关系。本研究筛选到CH03a09、CH02c06、CH04g12、CH02d12和CH05h05五对引物组合可区分除’Marc’与’Peluches’、‘美玉’与‘常绿2号’外的所有供试材料3、从78对枇杷多态性SSR引物中,筛选出55对在大五星多倍体枇杷株系中有多态性的引物,用于10个株系的基因型分析结果显示,55对引物共扩增出135个等位基因,其中CH01h02-222 bp为大五星三倍体特有的等位基因。引物CH01h02在A332中扩增出222 bp、199 bp和195 bp三个等位基因；引物CH04c06在A332中扩增出214 bp、195 bp和185 bp三个等位基因,在A368和A379两个株系中扩增出217 bp、195 bp和185 bp三个等位基因；NZ02b01在株系A332中扩增出270 bp、266 bp和246 bp三个等位基因,引物Hi15h12在A322中扩增出238 bp、236 bp和230 bp三个等位基因。所有三倍体株系与二倍体相比,都出现了新的等位基因,表明供试的三倍体株系形成过程中可能都有外源基因的渗入。10个株系完全区分开,各株系间SM相似性系数最高的是A2x与A313,相似性系数为0.926；最低的是A313与A332,相似性系数为0.496。主成分分析将10个株系分成三组,第Ⅰ组包括A484、A376、A379和A368四个株系,A35、A322和A332三个株系构成了第Ⅱ组,第Ⅲ组由A2x、A313和A484三个株系组成,揭示SSR可用于天然枇杷三倍体的遗传多样性分析,并能鉴定部分三倍体株系。更多还原

【Abstract】 Loquat [Eriobotrya japonica (Thunb) Lindl.], a rare and specialty fruits of subtropical areas, originated in China and has been cultivated for a Long history, with high economic value. Zhejiang and Jiangsu provinces are the main known producing areas of loquat, Also belong to highly evolved type of cultivation area, a lot of varieties and cultivars were developed. Therefore, study on the genetic diversity and phylogenetic relationship of Chinese main loquat germplasm resources based on the local cultivars and types of loquat cultivars in these areas, will has important guiding significance for innovative use and effective protection of loquat germplasm.In this study, a total of 54 cultivars or types were used, including main cultivars or types of loquat collected from Zhejiang and Jiangsu provinces, and cultivars of Fujian and Sichuan, two wild species E. prinoides var. daduheensis and E. Prinodides also included, and 110 located apple SSR markers were screened for genetic analysis of loquat SSR primers. The loquat microsatellite system was established to test for the genetic diversity and cultivar identification of 54 materials, genetic diversity among natural triploid strains of ’Dawuxing’ loquat also studied, and the primers that can be used for identification of natural triploid loquat were screened, the main results as follows:1. The SSR system was estabilished with eight cultivars as ’Jidanhong’,’Jiajiao’,’Changhong’, ’Tongpi’,’Dawuxing’,’Zaohuang’,’Dahongpao’ and ’Dameiguihongpao’. Seventy eight pairs of polymorphism primers were screened from 110 ones, the polymorphism ratio was 71 percent, and the appropriate annealing tempretures of these 78 were optimized with gradient PCR.2. Four markers (AT000400, CH01d03, CH01h02, and CH03c02) were judged to be multi-locus and not used for genetic diversity analysis. The 39 polymorphic SSR markers gave a total of 155 alleles, two to seven alleles per marker with an average of 3.38. The effective number of alleles varied from 1.04 for CH01f02 to 4.01 for CH03a09, with an average of 2.21. The Shannon index, as a measure of gene diversity, ranged from 0.10 for CH01f02 to 1.49 for CH03a09, with an average of 0.84. The observed heterozygosity ranged from approximately zero for CH01f02 to 0.83 for Hi08a04, with a mean of 0.47. Similar values were calculated for experted heterozygosity, the highest was 0.76 with CH03a09 and CH02d12, the lowest was 0.04 of CH01f02, ith a mean of 0.50. The fixation index ranged from-0.70 for Hi08a04 to 1.00 for CH01f02, with an average of 0.07. All these indicated that the genetic diversity of seleted materials was abundant. In the dendogram constructed from UPGMA cluster analysis of the similarity matrix, with 155 SSR alleles, the accessions are clustered in two groups, the commonly cultivated loquats and the two wild species at a 0.48 threshold of SM coefficient. The cultivated loquat accessions were further subdivided into three subgroups, at a threshold of 0.723, and generally reflected their geographic origin. Subgroup A included all. local accessions from the geographically close Zhejiang and Jiangsu provinces, being temperate zone accessions, plus the’Longquan No.l’from Sichuan and ’Changhong’ from Fujian, and the two Spanish cultivars. Subgroup B included the subtropical Fujian cultivars (’Zaozhong No.6’, ’Jiefangzhong’, ’Xiangzhong’, ’Taicheng No.4’ and ’Baili’), but also the Sichuan cultivar ’Jinfeng’ and the Japanese cultivar ’Moriowase’. The third subgroup included only one cultivar: ’Dawuxing’ of Sichuan. The clusters thus generally reflected the geographic origin of their members. PCA analyses, carried out using the similarity matrices for the 39 SSR markers, confirmed the UPGMA cluster analysis. The two wild species were separated from the cultivated accessions, and the cultivated accessions were classified in four groups, mainly accordingto their geographical distribution. All the Zhejiang accessions, ten Jiangsu accessions, the Japanese cultivar’Moriowase’, and two Spanish cultivars,’Marc’and’Peluches’, were in groupⅠ. The 15 Jiangsu accessions clustered in groupⅡ, all the Fujian cultivars in groupⅢ, and’Dawuxing’ and ’Longquan No.l’, the two most commonly cultivated varieties in Sichuan province, in groupⅣ. Thirty nine SSR markers distinguished all accessions except four cultivars:the two Spanish cultivars ’Marc’ and ’Peluches’ could not be distinguished from each other, and ’Meiyu’ could not be distinguished from ’Changlv No.2’. The scores of these markers confirmed pedigrees, such as that of’Zaozhong No.6’(a ’Jiefangzhong’×’Moriowase’),’Changlv No.5’(a ’Baiyu’×’Tianzhong’), and ’Xiangzhong’. Based on the number of effective alleles, we selected a set of five SSR markers (CH03a09, CH02c06, CH04g12, CH02d12, and CH05h05) able to distinguish all the accessions except bud sports.3. Fifty five pairs of polymorphism primers were screened from 78 ones, gave a total 135 alleles with ten strains of’Dawuxing’. The allele with 222 base pairs of CH01h02 was the specific one of the triploid. Three alleles of 222 bp,199 bp and 195 bp were gave with primer CH01h02 at strain A332, three alleles of 214bp,195 bp and 185 bp with primer CH04c06, and another three of 270 bp,266 bp and 246 bp with primer NZ02b01. Similar alleles of 217 bp,195 bp and 185 bp with primer CH04c06 at strain A368 and A379, also 238 bp,236 bp and 230 bp with primer Hil5hl2 at strain A322. New alleles emerged as compared diploid and each one of triploid strains, indicating foreign genes maybe introgressed along with the formation progress of triploid strains. All ten strains completely Distinguished from each other, the highest SM similarity coefficient was between A2x and A313, with 0.926, and the contrast one was between A313 and A332, with the similarity coefficient of 0.496. Principal component analysis divided 10 strains into three groups, GroupⅠ, including the A484, A376, A379 and A368 of four lines, A35, A322 and A332 are three lines constitute the groupⅡ, groupⅢconsists of A2x, A313 and A484. All these indicated that SSR can be used to reveal the genetic diversity of natural triploid loquat, and to identify some of triploid strains.更多还原